Lymphocyte development is regulated by a variety of cellular cues, many of which emanate from cell surface receptors. All developing lymphocytes must assemble genes encoding the different chains of the B cell receptor (BCR), or T cell receptor (TCR) and express a functional, non-auto-reactive, antigen receptor to exit the thymus (T cells) or bone marrow (B cells) and enter the periphery. Antigen receptor gene assembly occurs through the process of V(D)J recombination, which is initiated when the RAG proteins introduce DNA double strand breaks (DSBs) at V, D and J gene segments in antigen receptor loci. These DSBs are processed and joined by the proteins of the NHEJ pathway of DSB repair, appending the V, D and J gene segments to complete the second exon of antigen receptor genes. DSBs generated by genotoxic agents (such as ionizing radiation) activate cellular DNA damage responses including repair pathways and pathways that promote cell cycle arrest and cell death if the DSBs persist unrepaired. It has been unclear whether physiologic DNA breaks such as RAG DSBs also activate broad DNA damage responses or whether they are prevented from doing so unless they persist un-repaired, thus posing a hazard to the cell. Here, we show that in developing lymphocytes, RAG DSBs activate Atm, a kinase that is a major transducer of DNA damage response signals, and that this promotes a broad genetic program by engaging several transcriptional pathways including STAT3, Foxo1 and NF:B. Several of the genes regulated by RAG DSBs encode proteins that function in canonical DNA damage responses. Surprisingly however, a large fraction has no known function in DNA damage responses. Rather, the genes encode proteins that function in processes that are important for normal lymphocyte development. From this, we hypothesize that RAG DSBs provide signals that regulate developmental processes that are at a minimum, important for developing lymphocytes assembling antigen receptor genes. Here, we will test this hypothesis, elucidate the mechanisms by which RAG DSBs activate transcriptional pathways in developing lymphocytes and determine how the function of these pathways is integrated with other lymphocyte developmental signals. PUBLIC HEALTH RELEVANCE: All developing lymphocytes generate DNA double strand breaks (DSBs) as intermediates in the generation of complete antigen receptor genes. Here, we demonstrate that these DSBs activate a genetic program that includes genes encoding proteins with broad cellular functions including processes that are important for normal lymphocyte development. The goal of this project is to understand how these DSBs activate different transcription pathways and how the resulting genetic program participates in lymphocyte development. These studies will have a broader relevance to our understanding of lymphocyte development and the cellular responses to DNA damage.